The long term objective of this proposal is to study the function of keratin polypeptides 8 and 18 (K8/18). These proteins are the cytoplasmic intermediate filaments (IF) that are selectively expressed in "simple" type epithelia, such as in the gastrointestinal tract, liver and exocrine pancreas. IF are quite diverse structurally, and this diversity is reflected by expression in a tissue and differentiation- state specific manner. The function of IF remains unknown, however, [three] skin diseases were recently shown to have point mutations in IF, thereby suggesting that one function, at least for the epidermal keratins, is to provide cells with mechanical integrity. Most of IF structural diversity resides in the N-terminal "head" and C-terminal "tail" domains which contain most of the phosphorylation (phos) and recently identified glycosylation (gly). Our hypothesis is that K8/18 phos and gly are likely to be important functionally. This is based on their dynamic nature, their presence on distinct K8 and K18 molecules, their localization (in K18) within the structurally diverse N-terminal portion of the molecule, and the association of changes in phos with changes in filament organization during mitosis and in IF-containing inclusion bodies of several diseases (e.g. Mallory bodies of alcoholic liver disease). The overall specific objective of this proposal is the detailed study of K8/18 phos and gly. We plan to identify the specific sites of phos/gly using two approaches: a biochemical approach of selective proteolysis, mass spectrometry and microsequencing; and a molecular approach using [as a first step] expression of serine/threonine mutants in a baculovirus- insect cell system. The effect of specific phos/gly mutations on filament assembly [and disassembly, in vitro, or in cell transfectants during different stages of the cell cycle] will then be studied. Constructs expressing different parts of K8/18 will also be obtained to examine the structural requirements of K8/18 that dictate the level and nature of phos/gly. Our proposed studies should form the necessary basis for carrying out further molecular and genetic studies to elucidate the function of phos/gly in K8/18, and in turn the function of these proteins.